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Biosynthetic ganciclovir triphosphate: Its isolation and characterization from ganciclovir-treated herpes simplex thymidine kinase-transduced murine cells

  1. Author:
    Agbaria, R.
    Candotti, F.
    Kelley, J. A.
    Hao, Z.
    Johns, D. G.
    Cooney, D. A.
    Blaese, R. M.
    Ford, H.

  2. Author Address

    NHGRI, NIH, Bethesda, MD 20892 USA. NHGRI, NIH, Bethesda, MD 20892 USA. Ben Gurion Univ Negev, Fac Hlth Sci, Dept Clin Pharmacol, Beer Sheva, Israel. NCI, Ctr Canc Res, Med Chem Lab, Frederick, MD 21702 USA. Ford H NHGRI, NIH, Bethesda, MD 20892 USA.
    1. Year: 2001
  2. Journal: Biochemical and Biophysical Research Communications
    1. 289
    2. 2
    3. Pages: 525-530
  4. Type of Article: Article
  1. Abstract:

    A method is described for the preparation of ganciclovir triphosphate (GCV-TP) using murine colon cancer cells (MC38) transduced with the herpes simplex virus-thymidine kinase (MC38/HSV-tk). Murine cells transduced with viral-tk contain required viral and host enzymes needed for complete cellular synthesis of this potent antiviral metabolite. Dose response studies showed optimal intracellular levels of GCV-TP occurred after exposure of MC38/HSV-tk cells to 300 muM ganciclovir for 24 h producing 7.5 nmol GCV-TP/10(6) cells. This reflects cellular accumulation of GCV-TP to levels 25-fold greater than the medium concentration of parent drug. A simple isolation scheme included methanolic extraction and anion-exchange chromatography to recover the target triphosphate. Mass spectral analysis and selective enzyme degradation provided structural confirmation of the purified product. Biological activity of the purified GCV-TP was demonstrated by competitive inhibition experiments using human DNA polymerase a and HSV DNA polymerase that showed substantially greater sensitivity for the viral polymerase in agreement with previous reports. The GCV-TP obtained was further used to enzymatically prepare GCV mono- and diphosphate in high yield. This method provides an easily scalable means of preparing milligram amounts of the triphosphates of pharmacologically active acyclic nucleosides like ganciclovir.

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